tizoxanide  (Tokyo Chemical Industry)

Journal: Cell Reports Medicine

Article Title: Mitochondrial uncouplers inhibit oncogenic E2F1 activity and prostate cancer growth

doi: 10.1016/j.xcrm.2024.101890

Figure Lengend Snippet: Tizoxanide is a mitochondrial uncoupler that inhibits SKP2 expression and the growth of prostate cancer cells (A) Chemical structures of nitazoxanide (NTZ) and tizoxanide (TIZ). (B) PC3 cells preloaded with TMRE were treated with indicated compounds, and fluorescence intensity was measured. (C) PC3 cells were sequentially treated with oligomycin A, TIZ (or FCCP), and rotenone plus antimycin. Real-time oxygen consumption rates (OCRs) were measured with Seahorse XF. (D) Cells were treated with TIZ for 16 h, and the SKP2 mRNA levels were quantified by qRT-PCR. (E and F) Indicated cells were treated with TIZ for western blotting for SKP2 (E) and p27 (F) expression. (G) Cells treated with TIZ for 16 h were subjected to cell cycle analysis. (H and I) Cells treated with TIZ for 48 h were subjected to cell cycle analysis for subG0/G1 populations (H) or western blotting for cleaved PARP (cl-PARP) and cleaved caspase-3 (cl-Casp3) expression (I). Fl-PARP, full-length PARP. (J) Indicated cells treated with TIZ for 4 days were subjected to MTT assays. The data presented in (C) are mean ± SEM ( n = 6); all other data are presented as mean ± SD ( n = 3 or 4). ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; Student’s t test. See also Figure S2 .

Article Snippet: Tizoxanide , Cayman Chemical , cat#13693.

Techniques: Expressing, Fluorescence, Quantitative RT-PCR, Western Blot, Cell Cycle Assay

Journal: Cell Reports Medicine

Article Title: Mitochondrial uncouplers inhibit oncogenic E2F1 activity and prostate cancer growth

doi: 10.1016/j.xcrm.2024.101890

Figure Lengend Snippet: NTZ analogs lacking mitochondrial uncoupling activity fail to inhibit SKP2 expression and prostate cancer growth (A) Chemical structures of the investigated NTZ analogs. (B) PC3 cells preloaded with TMRE were treated with 20 μM of NTZ analogs, and fluorescence intensity was measured. (C) OCR was determined after treatments with 20 μM of NTZ analogs. (D) PC3 cells preloaded with TMRE were treated with 5 μM of NTZ analogs for fluorescence measurements. (E) PC3 cells treated with 20 μM of TIZ or analogs were subjected to western blotting. (F) PC3 cells treated with indicated TIZ or analogs were subjected to MTT assays. (G) The mitochondrial uncoupling activities (measured by %RFU at 5 μM) and IC 50 values of TIZ and its analogs were plotted. Spearman’s correlation coefficient was calculated with GraphPad Prism. The data presented in (C) are mean ± SEM ( n = 6); all other data are presented as mean, or mean ± SD ( n = 3).

Article Snippet: Tizoxanide , Cayman Chemical , cat#13693.

Techniques: Activity Assay, Expressing, Fluorescence, Western Blot

Journal: Cell Reports Medicine

Article Title: Mitochondrial uncouplers inhibit oncogenic E2F1 activity and prostate cancer growth

doi: 10.1016/j.xcrm.2024.101890

Figure Lengend Snippet: Mitochondrial uncouplers inhibit oncogenic E2F1 activity in prostate cancer cells (A) A Venn diagram shows the number of genes whose expression was altered by TIZ and/or MAL in C4-2 cells. (B and C) GSEA of genes downregulated by both TIZ and MAL shows E2F/E2F1 motifs (B) and E2F1 target genes (C) were enriched. (D) A heatmap shows changes in the expression of representative E2F1 target genes regulated by TIZ and MAL. Genes involved in DNA synthesis are colored. (E and F) Prostate cancer cells treated with TIZ for 16 h were subjected to qRT-PCR (E) or western blotting (F) for E2F1 expression. (G) C4-2 cells treated with TIZ for 16 h were subjected to qRT-PCR assays for expression of E2F1 target genes. (H) E2F1-KO PC3 clones generated by CRISPR-Cas9 were treated with or without TIZ for 16 h, and then subjected to qRT-PCR. The mRNA levels of indicated genes in TIZ-treated cells relative to the levels in respective DMSO-treated cells were calculated and presented in the graph. (I) The genes downregulated by TIZ or MAL were analyzed with GSEA, and enriched Reactome gene sets are displayed. NES, normalized enrichment score. (J) Heatmap shows lipogenic genes downregulated by both TIZ and MAL. (K) Indicated cells treated with TIZ were subjected to EdU staining, and EdU + cells were quantified. (L) PC3 cells treated with or without TIZ were stained with oil red O. The stains were extracted with isopropanol, and absorbance was measured at 510 nm (M and N) E2F1-KO cells were treated with 20 μM of TIZ for EdU staining (M) or oil red O staining (N). (O) E2F1-KO cells treated with or without 20 μM of TIZ were subjected to qRT-PCR assays for lipogenic gene expression. The data are presented as mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Student’s t test. See also Figure S3 .

Article Snippet: Tizoxanide , Cayman Chemical , cat#13693.

Techniques: Activity Assay, Expressing, DNA Synthesis, Quantitative RT-PCR, Western Blot, Clone Assay, Generated, CRISPR, Staining, Gene Expression

Journal: Cell Reports Medicine

Article Title: Mitochondrial uncouplers inhibit oncogenic E2F1 activity and prostate cancer growth

doi: 10.1016/j.xcrm.2024.101890

Figure Lengend Snippet: TIZ inhibits Rb phosphorylation while downregulating cyclin D1 levels by activating p38 (A and B) Indicated prostate cancer cells treated with TIZ for 16 h were subjected to western blotting to determine the Rb phosphorylation levels. (C) PC3 and C4-2 cells carrying an inducible sgRb construct were induced by doxycycline (DOX), treated with or without 20 μM of TIZ for 16 h, and subjected to western blotting. (D) The mRNA levels of indicated E2F target genes were determined by qRT-PCR in PC3 cells with or without DOX-induced Rb knockdown. The data are presented as mean ± SD. ∗∗, p < 0.01; ∗∗∗, p < 0.001; Student’s t test. (E and F) Indicated cells treated with TIZ for 16 h were subjected to western blotting. (G) PC3 cells infected with lentiviruses expressing CCND1, T286A, or empty vector (EV) were treated with TIZ for 16 h and subjected to western blotting. The intensities of p-Rb bands and E2F1/SKP2 bands were quantified using ImageJ and normalized to that of the total Rb and β-actin, respectively, and indicated under corresponding blots. (H) PC3 cells treated with or without TIZ in the presence/absence of 5 μM of MG132 were subjected to western blotting. (I) PC3 cells were treated with 20 μM of TIZ for different time for western blotting. (J) Indicated cells treated with TIZ for 16 h were subjected to western blotting. (K) PC3 cells treated with 20 μM of TIZ in the presence or absence of 20 μM of SB203580 were subjected to western blotting. The intensities of p -cyclin D1 were quantified and normalized to that of respective cyclin D1 bands and indicated under the p -cyclin D1 blot. (L) PC3 cells treated with TIZ for 16 h in the presence/absence of 20 μM of SB203580 were subjected to western blotting. (M and N) PC3 cells infected with lentiviruses expressing p38-specific shRNA (shp38) or scramble shRNA (shScr) were treated with 20 μM of TIZ for different time (M), or treated with different concentrations of TIZ for 16 h (N), and subjected to western blotting. SE, short exposure; LE, longer exposure. The numbers under the p -cyclin D1 bands were the relative intensities of p -cyclin D1 bands after normalized to that of the respective cyclin D1 bands. See also Figure S4 .

Article Snippet: Tizoxanide , Cayman Chemical , cat#13693.

Techniques: Phospho-proteomics, Western Blot, Construct, Quantitative RT-PCR, Knockdown, Infection, Expressing, Plasmid Preparation, shRNA

Journal: Cell Reports Medicine

Article Title: Mitochondrial uncouplers inhibit oncogenic E2F1 activity and prostate cancer growth

doi: 10.1016/j.xcrm.2024.101890

Figure Lengend Snippet: TIZ activates the AMPK-p38 pathway to decrease the cyclin D1 level and inhibit E2F1 activity (A) OXPHOS uncoupling can activate two pathways converging on an inhibition of mTORC1 activity. (B) Indicated cells were lysed, and intracellular ATP levels were determined. (C and D) PC3 cells were treated with TIZ for 16 h and subjected to ATP rate assays using Seahorse XF. (E–G) Indicated cells treated with TIZ for 16 h were subjected to western blotting. (H) Indicated cells treated with TIZ for 16 h were incubated with puromycin for 30 min, and then lysed for western blotting using an anti-puromycin antibody (α-puro). Newly synthesized proteins were labeled by puromycin. The blots were also stained with Ponceau S (PonS) to confirm equal loading. (I) PC3 cells were infected with lentiviruses expressing shTSC2 or a scramble shRNA (shScr), and then treated with TIZ for western blotting. (J) PC3 cells infected with lentiviruses expressing shATF4 or shLuc were treated with TIZ and subjected to western blotting. (K) Indicated cells treated with 20 μM of TIZ were subjected to qRT-PCR assays. (L and M) AMPK α1/α2 double knockout (dKO) PC3 cells generated by CRISPR-Cas9 were treated with TIZ for 16 h and subjected to western blotting (M) or qRT-PCR (M). (N) A diagram shows that uncouplers activate the AMPK-p38 pathway to inhibit E2F1 activity. The data are presented as mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; Student’s t test. See also Figures S5 and .

Article Snippet: Tizoxanide , Cayman Chemical , cat#13693.

Techniques: Activity Assay, Inhibition, Western Blot, Incubation, Synthesized, Labeling, Staining, Infection, Expressing, shRNA, Quantitative RT-PCR, Double Knockout, Generated, CRISPR

Journal: Cell Reports Medicine

Article Title: Mitochondrial uncouplers inhibit oncogenic E2F1 activity and prostate cancer growth

doi: 10.1016/j.xcrm.2024.101890

Figure Lengend Snippet:

Article Snippet: Tizoxanide , Cayman Chemical , cat#13693.

Techniques: Recombinant, cDNA Synthesis, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: Nitazoxanide controls virus viability through its impact on membrane bioenergetics

doi: 10.1038/s41598-024-78694-8

Figure Lengend Snippet: The three thiazolides: Nitazoxanide, tizoxanide, RM4848 and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP).

Article Snippet: Thiazolides: Tizoxanide (dAcetyl nitazoxanide) and RM4848 were obtained from Romark Laboratories, tizoxanide will be abbreviated as TZ and RM4848 as RM hereafter.

Techniques:

Journal: Scientific Reports

Article Title: Nitazoxanide controls virus viability through its impact on membrane bioenergetics

doi: 10.1038/s41598-024-78694-8

Figure Lengend Snippet: Increased impact on OXPHOS and viral RNA release. The number of viral RNA copies in the supernatant of the virus producing cells (Phoenix-ECO GFP + +) was assessed by RTqPCR titration at 24 h posttreatment with TZ, RM or CCCP. The levels of impact on OXPHOS-OCR are indicated below the groups of histograms and are coded from white (0% impact on OXPHOS-OCR) to black (100% impact and full inhibition by oligomycin). The number of data points reveals number of independent experiments and histograms represent mean value ± SD. ( a ) The reference data points are as follows: hexagons, control (no addition); diamonds, vehicle (DMSO); squares, reference uncoupler (CCCP); and downward triangles, oligomycin. Control, vehicle and oligomycin were references included in each single experiment (N = 6). Thiazolides are indicated by black symbols; circles, tizoxanide and triangles, RM4848. All impact levels with uncouplers (TZ, RM and CCCP) could not be included in each single experiment and N ranged from 2 to 4. ( b ) To improve comparison between the different experiments, the viral RNA copy number was expressed as the ratio to control in the same experiment and values obtained with the uncouplers (TZ, RM, CCCP) were pooled according to their % impact on OXPHOS-OCR. Statistical significance vs vehicle is shown as: * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001, for comparison with vehicle (DMSO).

Article Snippet: Thiazolides: Tizoxanide (dAcetyl nitazoxanide) and RM4848 were obtained from Romark Laboratories, tizoxanide will be abbreviated as TZ and RM4848 as RM hereafter.

Techniques: Virus, Titration, Inhibition, Control, Comparison

Journal: Scientific Reports

Article Title: Nitazoxanide controls virus viability through its impact on membrane bioenergetics

doi: 10.1038/s41598-024-78694-8

Figure Lengend Snippet: Increased impact on OXPHOS efficiency and the infectivity of released viruses. Infectivity was evaluated from the percentage of NIH3T3 positive for expression of the green fluorescent protein (see M & M section). Data points from independent experiments are shown and histograms represent mean value ± SD. ( a ) Measurement of infectivity of virus particles, the reference data points are as follows: hexagons, control (no addition); diamonds, vehicle (DMSO); squares, reference uncoupler (CCCP); downward triangles, oligomycin (100% impact). Thiazolides are indicated by black symbols; circles, TZ and triangles, RM. Experiment with 3 mM shown examined supernatants used previously for viral RNA release (Fig. ). Statistical significance is shown with regard to vehicle. ( b ) Expression of infectivity in relative units with uncouplers grouped according to level on OXPHOS-OCR (as in Fig. b) more experiments with 3 mM are included (see text). Statistical significance is shown as: * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001, for comparison with vehicle.

Article Snippet: Thiazolides: Tizoxanide (dAcetyl nitazoxanide) and RM4848 were obtained from Romark Laboratories, tizoxanide will be abbreviated as TZ and RM4848 as RM hereafter.

Techniques: Infection, Expressing, Virus, Control, Comparison

Journal: Scientific Reports

Article Title: Nitazoxanide controls virus viability through its impact on membrane bioenergetics

doi: 10.1038/s41598-024-78694-8

Figure Lengend Snippet: Mitochondrial uncoupling by TZ, RM and CCCP. ATP production (Oxphos). ( a – c ) Dose response to thiazolides and to CCCP. The values of the OCR (solid line) and mitochondrial membrane potential (milliVolts) are shown starting from oligomycin and the nonphosphorylating rate (X = 0) and with increasing concentrations of TZ (a,c,e), RM ( b , d , f ) or CCCP ( g ) at different concentrations, a-d and g mean value ± SD of three or more independent experiments (mitochondrial preparations). Experiments comparing the effects of thiazolides in the presence (gray lines) or absence (black lines) of CAT ( c , d ) or CSA ( e , f one experiment only) are shown without symbols for clarity. ( h ) Products of the OCR and membrane potential (in mV) for the three uncouplers; the symbols corresponding to ( a , b , g ) and the TZ and CCCP values are shown up to the maximal value and not for higher concentrations. This product could be converted into power in Watts (see Materials and Methods section), and a Y ordinate of 100 in D indicates 116 µW/mg protein (116 W/kg protein) dissipated by the proton circuit (inset in h ).

Article Snippet: Thiazolides: Tizoxanide (dAcetyl nitazoxanide) and RM4848 were obtained from Romark Laboratories, tizoxanide will be abbreviated as TZ and RM4848 as RM hereafter.

Techniques: Membrane

Journal: Scientific Reports

Article Title: Nitazoxanide controls virus viability through its impact on membrane bioenergetics

doi: 10.1038/s41598-024-78694-8

Figure Lengend Snippet: Mitochondrial ATP production (Oxphos). ( a ) Relationships between the oxygen consumption rate and the membrane potential in mitochondria at steady state. Individual gray diamonds show individual data points obtained with four independent mitochondrial preparations used for ATP measurement (controls in b – d ) “ P ” indicates the phosphorylating state (ADP present), while L indicates the absence of phosphorylation (oligomycin present). The intermediate data points (with error bars) correspond to the first (left) values shown in Fig. a,b,g excluding values when potential dropped or after maximal OCR hence five values with TZ (black circles) four with RM (black triangles) and three with CCCP (empty squares). ( b ) kinetic of phosphorylation of ADP into ATP in presence/absence of thiazolides, Y represents results for ATP sampling (see M&M section) with control (gray diamonds), TZ (black circles) or RM (empty triangles) it is expressed in relative units: 100% is the ATP produced by the respective control in five minutes, actual values are shown in supplemental a. A same dosage of TZ or RM was used in each of the four experiments but varied from one experiment to another to adjust the membrane potential signal to a value consistent with that observed in presence of ADP (and the concentrations used were therefore: 6, 10, 12 and 20 µM). Other experiments (Suppl. a) implied lower concentrations of thiazolides 3, 5, 6 and 5 µM respectively. ( c ) ATP/O ratio for the controls (CTRL) and in the presence of TZ or RM at the concentration used in b (High TZ, High RM) or with the lower concentrations (Low TZ, Low RM; individual values are shown with their mean (± sem). ( d ) Relationship between the ATP synthesis rate obtained from the ATP dosage (Y axis) and the decrease in the OCR caused by the ATP synthase inhibitor oligomycin: OXPHOS-OCR (X axis). Note that comparison between c and d must take into account that OXPHOS-OCR is expressed per oxygen molecule (O 2 ).

Article Snippet: Thiazolides: Tizoxanide (dAcetyl nitazoxanide) and RM4848 were obtained from Romark Laboratories, tizoxanide will be abbreviated as TZ and RM4848 as RM hereafter.

Techniques: Membrane, Phospho-proteomics, Sampling, Control, Produced, Concentration Assay, Comparison

Journal: Scientific Reports

Article Title: Nitazoxanide controls virus viability through its impact on membrane bioenergetics

doi: 10.1038/s41598-024-78694-8

Figure Lengend Snippet: The three thiazolides: Nitazoxanide, tizoxanide, RM4848 and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP).

Article Snippet: Thiazolides: Tizoxanide (dAcetyl nitazoxanide) and RM4848 were obtained from Romark Laboratories, tizoxanide will be abbreviated as TZ and RM4848 as RM hereafter.

Techniques:

Journal: Scientific Reports

Article Title: Nitazoxanide controls virus viability through its impact on membrane bioenergetics

doi: 10.1038/s41598-024-78694-8

Figure Lengend Snippet: Increased impact on OXPHOS and viral RNA release. The number of viral RNA copies in the supernatant of the virus producing cells (Phoenix-ECO GFP + +) was assessed by RTqPCR titration at 24 h posttreatment with TZ, RM or CCCP. The levels of impact on OXPHOS-OCR are indicated below the groups of histograms and are coded from white (0% impact on OXPHOS-OCR) to black (100% impact and full inhibition by oligomycin). The number of data points reveals number of independent experiments and histograms represent mean value ± SD. ( a ) The reference data points are as follows: hexagons, control (no addition); diamonds, vehicle (DMSO); squares, reference uncoupler (CCCP); and downward triangles, oligomycin. Control, vehicle and oligomycin were references included in each single experiment (N = 6). Thiazolides are indicated by black symbols; circles, tizoxanide and triangles, RM4848. All impact levels with uncouplers (TZ, RM and CCCP) could not be included in each single experiment and N ranged from 2 to 4. ( b ) To improve comparison between the different experiments, the viral RNA copy number was expressed as the ratio to control in the same experiment and values obtained with the uncouplers (TZ, RM, CCCP) were pooled according to their % impact on OXPHOS-OCR. Statistical significance vs vehicle is shown as: * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001, for comparison with vehicle (DMSO).

Article Snippet: Thiazolides: Tizoxanide (dAcetyl nitazoxanide) and RM4848 were obtained from Romark Laboratories, tizoxanide will be abbreviated as TZ and RM4848 as RM hereafter.

Techniques: Virus, Titration, Inhibition, Control, Comparison

Journal: bioRxiv

Article Title: Pseudohypoxia-stabilized HIF2⍺ transcriptionally inhibits MNRR1, a druggable target in MELAS

doi: 10.1101/2024.10.07.617011

Figure Lengend Snippet: A: Oxygen consumption measured from MELAS cybrid cells treated with vehicle (DMSO) or tizoxanide (10 μM) for 48 h (n=4 biological replicates, error bars represent SE). B: HaeIII restriction enzyme digestion of a PCR-amplified fragment of mtDNA harboring the m.3243A>G mutation from DW7 cells treated with vehicle (DMSO) or tizoxanide (10 μM) for 144 h. The table shows n=4 biological replicates as mean ± SD. C : Genes identified using qPCR of MELAS cybrid cells stably overexpressing EV or MNRR1. D: MtDNA levels are shown relative to nuclear DNA (nDNA) (GAPDH) (n=5 biological replicates, error bars represent SE). E: Equal amounts of MELAS cybrid cell lysates, treated with vehicle (DMSO) or tizoxanide (10 μM) for 48 h, were separated on an SDS-PAGE gel and probed for PGC1α, PINK1, LC3 A/B, MNRR1, and Tubulin. F: Equal numbers of MELAS cells were treated as in E , separated on an SDS-PAGE gel, and probed for phospho-ubiquitin (S65) levels. Tubulin was probed as a loading control. In all figures, * indicates p<0.05, ** indicates p<0.005.

Article Snippet: Tizoxanide, nitazoxanide, and genetisate were obtained from Selleckchem (Houston, TX), Phenyl-4-aminosalicylic acid was obtained from A2Bchem (San Diego, CA), 2-Amino 5-nitrothiazole was from Sigma, and the remaining compounds (4-Aminobenzanilide, 4-Amino salicylic acid, benzanilide, phenyl benzoate, and phenyl salicylic acid) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Amplification, Mutagenesis, Stable Transfection, SDS Page, Control

Journal: bioRxiv

Article Title: Pseudohypoxia-stabilized HIF2⍺ transcriptionally inhibits MNRR1, a druggable target in MELAS

doi: 10.1101/2024.10.07.617011

Figure Lengend Snippet: A: Equal amounts of lysates from MELAS patient fibroblast (MF 1, 2, or 3) lysates treated with vehicle (DMSO) or tizoxanide (10 μM) for 48 h were separated on an SDS-PAGE gel and probed for LC3B, PGC1α, MNRR1, MTCO2, TOM20, and phospho-ubiquitin (S65) levels. GAPDH or Tubulin was probed as a loading control. B: Oxygen consumption measured from MELAS patient fibroblasts treated with vehicle (DMSO) or tizoxanide (10 μM) for 48 h (n=4 biological replicates, mean ± SE). C: Mitochondrial ATP rate measured in MELAS patient fibroblasts treated with vehicle (DMSO) or tizoxanide (10 μM) for 48 h (n=4 biological replicates, mean ± SE). D: Heatmap representation of ROS and TNFa transcript levels from MELAS patient fibroblasts treated with vehicle (DMSO), LPS (DMSO + 500 ng/ mL LPS), or LPS plus tizoxanide (10 μM tizoxanide + 500 ng/mL LPS) for 24 h (n=4 biological replicates). In all figures, * indicates p<0.05, ** indicates p<0.005.

Article Snippet: Tizoxanide, nitazoxanide, and genetisate were obtained from Selleckchem (Houston, TX), Phenyl-4-aminosalicylic acid was obtained from A2Bchem (San Diego, CA), 2-Amino 5-nitrothiazole was from Sigma, and the remaining compounds (4-Aminobenzanilide, 4-Amino salicylic acid, benzanilide, phenyl benzoate, and phenyl salicylic acid) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: SDS Page, Control

Journal: bioRxiv

Article Title: Pseudohypoxia-stabilized HIF2⍺ transcriptionally inhibits MNRR1, a druggable target in MELAS

doi: 10.1101/2024.10.07.617011

Figure Lengend Snippet: A: Schematic representation of deleted regions of the MNRR1 promoter. B: Dual luciferase reporter assay using the MNRR1 promoter deletions in cells treated with vehicle (DMSO), nitazoxanide, or tizoxanide (10 μM) for 24 h (n=4 biological replicates). C: DNA sequence of region of 800-952 bp in the MNRR1 promoter highlighting binding sites of the transcription factors shown. D: Transcript levels of HIF2α and HIF1α in MELAS cybrid cells harboring different levels of heteroplasmy (0% to 100%). Data from . E: Protein levels of HIF2α and HIF1α in cybrid cells with 0% (CL9) and 70% MELAS heteroplasmy (DW7) In all figures, * indicates p<0.05, ** indicates p<0.005.

Article Snippet: Tizoxanide, nitazoxanide, and genetisate were obtained from Selleckchem (Houston, TX), Phenyl-4-aminosalicylic acid was obtained from A2Bchem (San Diego, CA), 2-Amino 5-nitrothiazole was from Sigma, and the remaining compounds (4-Aminobenzanilide, 4-Amino salicylic acid, benzanilide, phenyl benzoate, and phenyl salicylic acid) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Luciferase, Reporter Assay, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Pseudohypoxia-stabilized HIF2⍺ transcriptionally inhibits MNRR1, a druggable target in MELAS

doi: 10.1101/2024.10.07.617011

Figure Lengend Snippet: A: Above , Equal amounts of MELAS cell lysates, treated with vehicle (DMSO) or increasing amounts of tizoxanide (2.5 to 40 μM) for 24 h, were separated on an SDS-PAGE gel and probed for HIF2α, MNRR1, and Tubulin. Below, Protein quantification for MNRR1 and HIF2α relative to Tubulin. B: Dual luciferase reporter assay showing relative activation of MNRR1 -luciferase in MELAS cybrid cells overexpressing WT , Δ 801-952 , ΔORE (sequence in orange in ) or ΔHRE (sequence in red in ). C: Dual luciferase reporter assay showing relative activation of HRE -luciferase levels in cybrid cells with 0% (CL9) and 70% MELAS heteroplasmy (DW7). D : Dual luciferase reporter assay showing relative activation of MNRR1 -luciferase levels in MELAS cybrid cells with 0% heteroplasmy (CL9) overexpressing EV (empty vector) or HIF2α. E: Sequences in MNRR1 promoter highlighting the binding sites for RBPJκ (orange) and HIF2α (blue) on opposite strands of DNA. In all figures, * indicates p<0.05, ** indicates p<0.005.

Article Snippet: Tizoxanide, nitazoxanide, and genetisate were obtained from Selleckchem (Houston, TX), Phenyl-4-aminosalicylic acid was obtained from A2Bchem (San Diego, CA), 2-Amino 5-nitrothiazole was from Sigma, and the remaining compounds (4-Aminobenzanilide, 4-Amino salicylic acid, benzanilide, phenyl benzoate, and phenyl salicylic acid) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: SDS Page, Luciferase, Reporter Assay, Activation Assay, Sequencing, Plasmid Preparation, Binding Assay

Journal: bioRxiv

Article Title: Pseudohypoxia-stabilized HIF2⍺ transcriptionally inhibits MNRR1, a druggable target in MELAS

doi: 10.1101/2024.10.07.617011

Figure Lengend Snippet: A: Transcript levels of PHD1, 2, and 3 in MELAS cybrid cells harboring the levels of heteroplasmy shown (data from ). B: Protein levels of PHD3 in MELAS cybrid cells with 0% (CL9) and 70% heteroplasmy (DW7). GAPDH was probed as loading control. C: Equal amounts of MELAS cybrid cell lysates, treated with vehicle (DMSO), nitazoxanide, or tizoxanide (10 μM) for 24h, were separated on an SDS-PAGE gel and probed for PHD1, PHD3, MNRR1, and Tubulin. D: Oxygen consumption measured from MELAS cybrid cells overexpressing PHD1 or PHD3. (n=4 biological replicates, error bars show SE).

Article Snippet: Tizoxanide, nitazoxanide, and genetisate were obtained from Selleckchem (Houston, TX), Phenyl-4-aminosalicylic acid was obtained from A2Bchem (San Diego, CA), 2-Amino 5-nitrothiazole was from Sigma, and the remaining compounds (4-Aminobenzanilide, 4-Amino salicylic acid, benzanilide, phenyl benzoate, and phenyl salicylic acid) were from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Control, SDS Page